WO2023039405A1 - Méthodes d'utilisation d'inhibiteurs d'usp15 - Google Patents

Méthodes d'utilisation d'inhibiteurs d'usp15 Download PDF

Info

Publication number
WO2023039405A1
WO2023039405A1 PCT/US2022/076022 US2022076022W WO2023039405A1 WO 2023039405 A1 WO2023039405 A1 WO 2023039405A1 US 2022076022 W US2022076022 W US 2022076022W WO 2023039405 A1 WO2023039405 A1 WO 2023039405A1
Authority
WO
WIPO (PCT)
Prior art keywords
usp15
cells
subject
protein
inhibitor
Prior art date
Application number
PCT/US2022/076022
Other languages
English (en)
Inventor
Thang VAN NGUYEN
Original Assignee
The Curators Of The University Of Missouri
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Curators Of The University Of Missouri filed Critical The Curators Of The University Of Missouri
Publication of WO2023039405A1 publication Critical patent/WO2023039405A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/45—Non condensed piperidines, e.g. piperocaine having oxo groups directly attached to the heterocyclic ring, e.g. cycloheximide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/454—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. pimozide, domperidone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
    • A61K31/5513—1,4-Benzodiazepines, e.g. diazepam or clozapine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/69—Boron compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57426—Specifically defined cancers leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/44—Multiple drug resistance
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present disclosure provides compositions and methods related to USP15 inhibitors. More particularly, the present disclosure is directed to methods of regulating the CRL4 CRBN -USP15 pathway (previously referred to as the CRL4 CRBN -p97 pathway) and glutamine synthetase levels and methods of treating diseases such as cancer via administration of USP15 inhibitors.
  • Thalidomide was prescribed to pregnant women as a sedative to treat morning sickness in the late 1950s. It was withdrawn from the market in the early 1960s, when it was found to be a cause of severe birth defects. Recently, thalidomide and its immunomodulatory derivatives (IMiDs) lenalidomide and pomalidomide, have been used for the treatment of multiple myeloma (MM) and other hematologic malignancies. Other thalidomide analogs, now known as the Cereblon E3 ligase modulators (CELMoDs), including CC-122, CC-220 and CC-885, have been developed to target pathogenic proteins for degradation via a molecular glue mechanism.
  • IiDs immunomodulatory derivatives
  • CELMoDs Cereblon E3 ligase modulators
  • CC- 885 induces degradation of the translation termination factor GSPT1, a CRL4 CRBN neo-substrate.
  • CRBN is also a target for the development of proteolysis-targeting chimaera (PROTAC) technology, which relies on linking a drug that binds to a protein of interest to IMiDs.
  • PROTAC proteolysis-targeting chimaera
  • CRBN is required for the action of IMiDs, but CRBN protein levels do not correlate with intrinsic sensitivity or resistance to IMiDs in MM cell lines, suggesting that other factors play a critical role in regulating the mechanisms underlying sensitivity and/or resistance to IMiDs.
  • cereblon (CRBN) protein is a primary target of thalidomide teratogenicity and that CRBN is key target of antitumor activities, and the CRL4 CRBN -p97 pathway was only recently discovered.
  • IMDs immunomodulatory drugs
  • lenalidomide lenalidomide
  • pomalidomide thalidomide
  • MM myeloma
  • Cereblon (CRBN) protein is a direct target for thalidomide teratogenicity and antitumor activity of IMiDs.
  • the binding of IMiDs to CRBN promotes the recruitment of neo-substrates including Ikaros (IKZF1) and Aiolos (IKZF3) and casein kinase la (CKla), leading to their ubiquitylation and subsequent degradation.
  • IKZF1 Ikaros
  • IKZF3 casein kinase la
  • CRBN is required for antitumor activity of IMiDs, but its expression levels do not correlate with intrinsic sensitivity or resistance to IMiDs in MM cells, suggesting that another factor is essential for regulating the mechanisms of IMiD sensitivity and resistance. It was discovered the CRL4 CRBN -p97 pathway was required for degradation of GS and neo-substrates (such as GS, IKZF1, IKZF3, CKla, RNF166, GSPT1 and BRD4).
  • CRBN-based platforms have been used to develop small molecules to target pathogenic proteins for degradation via molecular glue and PROTAC mechanisms.
  • the most fundamental question in the field is how the CRL4 CRBN -p97 pathway manages to degrade target proteins.
  • DUBs Deubiquity dating enzymes
  • UCHs ubiquitin C-terminal hydrolases
  • USPs ubiquitin-specific proteases
  • OTUs ovarian tumor proteases
  • JAB1/MPN/MOV34 metalloenzymes JAB1/MPN/MOV34 metalloenzymes
  • USP15 High expression of USP15 was significantly correlated with poor survival rate within the pan-cancer patient cohort, representing a key feature of oncogene activity.
  • USP15 has been shown to deubiquitylate and stabilize protein substrates in different signaling pathways such as TGF-P, MDM2 and NF-KB. Since UsplS -1- mice were viable, targeting USP15 in cancer could achieve major advantages for an optimal therapeutic window. However, the molecular mechanism underlying the oncogenic activity of USP15 remains elusive.
  • Glutamine plays important roles in many cellular processes, including oxidative metabolism and ATP generation, biosynthesis of proteins, lipids and nucleic acids, as well as regulation of mTOR signaling pathway and autophagy.
  • Glutamine synthetase (GS) is the only enzyme that is capable of the de novo synthesis of glutamine and detoxifies glutamate and ammonia. Mutations and deregulation of GS have been linked to human diseases, including congenital glutamine deficiency, Alzheimer’s disease, and cancers in particular glioblastoma (GBM).
  • p97 acts downstream of CRL4 CRBN to promote disassembly of ubiquitylated GS subunits, which are subsequently degraded by the proteasome. It was also recently found that endogenous GS protein levels are negatively regulated by glutamine through a feedback loop involving CRL4 CRBN2 °. However, it remains unknown how cells sense extracellular glutamine levels to control GS stability in the CRL4 CRBN -p97 pathway.
  • VCP valosin-containing protein
  • p97 extracts ubiquity lated GS subunits from the decamer so that they can be degraded by the proteasome. Many important questions about this process remain unanswered. Two questions are how cells sense glutamine levels to control GS acetylation, and whether acetylation may be a general mechanism for targeting substrates to CRBN.
  • Both lysine 11 and lysine 14 on GS are acetylated, resulting in CRBN binding, CRBN-dependent ubiquitylation and subsequent degradation by the proteasome.
  • GS is the only endogenous substrate for CRBN has been well characterized.
  • understanding how CRBN recognizes acetylated substrates and how this is influenced by IMiDs could create new opportunities for IMiD therapy.
  • GS Hepatocellular carcinoma
  • CML chronic myelogenous leukemia
  • AML acute myelogenous leukemia
  • prostate express higher GS mRNA levels.
  • high GS expression may facilitate tumorigenesis, in that transgenic zebrafish, expressing an activated form of the Hippo pathway effector Yap 1 (YAP) that reprograms glutamine metabolism through activation of GS expression, develop enlarged livers and are prone to liver tumorigenesis.
  • YAP Hippo pathway effector Yap 1
  • the Hippo pathway can activate many other downstream target genes that may contribute to tumor development.
  • the in vivo function of GS in tumorigenesis remains to be determined.
  • One aspect of the disclosure is directed to a method of treating cancer in a subject in need thereof, the method comprising administering to the subject in need thereof a therapeutically effective amount of a USP15 inhibitor and at least one chemotherapeutic drug.
  • Another aspect of the disclosure is directed to methods of diagnosing and treating resistance to IMiD therapy in a subject with multiple myeloma undergoing IMiD therapy, the method comprising: a) measuring an amount of USP15 protein in the subject; b) comparing the amount of USP15 protein in the subject to a reference; c) diagnosing the subject with resistance to IMiD therapy if the amount of USP15 protein in the subject is higher than the reference; and d) administering a USP15 inhibitor to the subject.
  • a further aspect of the disclosure is directed to a method of regulating CRL4 CRBN - USP15 pathway and glutamine synthetase levels in a subject in need thereof, the method comprising: administering to the subject in need thereof a USP15 inhibitor.
  • FIG. 1A depicts a Western blot. 293FT cells were transfected with GS Myc and USP15 Flag . After 48 h, cell extracts were immunoprecipitated with anti-Flag antibody.
  • Fig. IB depicts a Western blot.
  • Cell extracts from 293 cells were immunoprecipitated with rabbit IgG control or USP15 antibodies, and then analyzed by immunoblotting (IB) with the indicated antibodies.
  • Fig. 1C depicts a Western blot.
  • Cell extracts from H1299 cells were immunoprecipitated with rabbit IgG control or USP15 antibodies, and then analyzed by immunoblotting (IB) with the indicated antibodies.
  • Fig. ID depicts a Western blot. MCF7 cells, starved of glutamine for 24 h, were treated with MG132 (10 pM) and/or 40 pM NSC632839, 20 pM PR-619 for 0.5 h, followed by 4 mM glutamine treatment for 2h. Total ubiquity lated proteins were purified using TUBE2-agarose, and then analyzed by IB with indicated antibodies. The relative ratio of ubiquity lated GS, GS(Ub)n to input GS, normalized to that of glutamine-treated cells in lane 2, are shown.
  • Fig. IE depicts a Western blot.
  • Cell extracts were analyzed by IB. The relative ratio of GS:Actin is shown.
  • Fig. IF depicts a Western blot.
  • Cell extracts were analyzed by IB. The relative ratio of GS: Actin is shown.
  • Fig. 1G depicts a Western blot. H1299 cells were treated with NSC632839 and 2 pM MLN4924 for 0.5 h, followed by 4 mM glutamine treatment for 8 h. Cell extracts were analyzed by IB. The relative ratio of GS: Actin is shown.
  • Fig. 1H depicts a Western blot. H1299 cells were treated with PR-619 and 2 pM MLN4924 for 0.5 h, followed by 4 mM glutamine treatment for 8 h. Cell extracts were analyzed by IB. The relative ratio of GS:Actin is shown.
  • Fig. 2A depicts a Western blot.
  • USP15 +!+ and USPIS -1- 293FT cells triplicate were cultured in 2 mM glutamine. Cell extracts were analyzed by IB. The relative ratio of GS : Actin is shown.
  • Fig. 2B depicts a Western blot. USP15 +l+ and USPIS -1- 293FT cells (triplicate) were cultured in different glutamine concentrations. Cell extracts were analyzed by IB. The relative ratio of GS: Actin is shown.
  • Fig. 2C depicts a Western blot.
  • USP15 +i+ and USPIS -1- 293FT cells triplicate were cultured in CHX. Cell extracts were analyzed by IB. The relative ratio of GS:Actm is shown.
  • FIG. 2D depicts a Western blot demonstrating rescue of endogenous GS protein levels in USPI5-KO cells.
  • USP15 +/+ and USPIS -1- 293FT cells were grown in complete medium, and then treated with 2 pM MLN4924 for 10 h.
  • Cell extracts were analyzed by IB with the indicated antibodies. The relative ratio of GS: Actin is shown.
  • Fig. 2E depicts a Western blot demonstrating rescue of endogenous GS protein levels in USP15-KO cells.
  • USP15 +/+ and USP15 ⁇ ! ⁇ 293FT cells were grown in complete medium, and then treated with 2 M MSO for 10 h. Cell extracts were analyzed by IB with the indicated antibodies. The relative ratio of GS:Actin is shown.
  • Fig. 2F depicts a Western blot demonstrating rescue of endogenous GS protein levels in USP15-KO cells.
  • USP15 +/+ and USP15 ⁇ ' ⁇ 293FT cells were staved of glutamine for 24 h, and then pre-treated with 2 pM MLN4924 or 2 mM MSO for 30 min, followed by 4 mM glutamine treatment for 8 h.
  • Cell extracts were analyzed by IB with the indicated antibodies. The relative ratio of GS: Actin is shown.
  • Fig. 2G depicts a Western blot. 293 cells stably expressing Dox-inducible shRNA targeting USP15 clone 1 (GSP75-shRNA TRIPZ_1) were induced with or without Dox (2 pg/ml) for 72 h. Cell extracts were analyzed by IB with the indicated antibodies. The relative ratio of GS: Actin is shown.
  • Fig. 2H depicts a Western blot. 293 cells stably expressing Dox-inducible shRNA targeting USPI5 clone 1 (US'P/5-shRNA TRIPZ_1) were induced with or without Dox (2 pg/ml) for 72 h. Cells were also grown in different glutamine concentrations. Cell extracts were analyzed by IB with the indicated antibodies. The relative ratio of GS:Actin is shown.
  • Fig. 21 depicts a Western blot. 293 cells stably expressing Dox-inducible shRNA targeting USPI5 clone 1 (GSP75-shRNA TRIPZ_1) were induced with or without Dox (2 pg/ml) for 72 h. Cells were also starved of glutamine for 24h, followed by 4 mM glutamine treatment. Cell extracts were analyzed by IB with the indicated antibodies. The relative ratio of GS:Actin is shown.
  • Fig. 2J depicts a Western blot showing validation of L4SP75-knockout (KO) 293FT cells via CRISPR/Cas9 gene editing.
  • Wild-type (WT) 293FT cells and USP15-KO clones 1 and 2 were validated by Western blot.
  • USP15-KO2 293FT cells were chosen for further analysis.
  • Fig. 2K depicts a Western blot showing. 293 cells stably expressing Dox-inducible shRNA targeting USP15 clone 1 (OSP/J-shRNA TRIPZ_1) were induced with or without Dox (2 pg/ml) for 72 h. Cells were also treated with cycloheximide (CHX, 100 pg/ml). Cell extracts were analyzed by IB with the indicated antibodies. The relative ratio of GS:Actin is shown.
  • Fig. 2L depicts the relative GS mRNA level as analyzed by quantitative RT-PCR.
  • FIG. 3A depicts a Western blot. Glutamine-starved USP15 +/+ and USPI5 ⁇ 293FT cells were treated with 10 pM CB-5083 for 30 min, followed by 4 m glutamine treatment for 3 h. The relative ratio of ubiquitylated GS, GS(Ub)n to input GS is shown.
  • Fig. 3B depicts a Western blot.
  • Total ubiquitylated proteins were purified using TUBE2-agarose.
  • the relative ratio of ubiquitylated GS, GS(Ub)n to input GS is shown.
  • Fig. 3C depicts a Western blot. USP15 ⁇ ' ⁇ 293FT cells were transfected with control plasmid, wild-type WT USP15 Flag or C298A-USP15 Flag mutant plasmid.
  • Fig. 3D depicts an in vitro deubiquitylation assay.
  • USP15 ⁇ 293FT cells starved off glutamine for 24 h, were pre-treated with CB-5083 for 0.5 h, followed by 4 mM glutamine treatment for 4 h.
  • Total ubiquitylated proteins were affinity-purified using TUBE2-agarose beads, which were subsequently treated with or without recombinant USP15 (rUSP15) protein at 37°C for 1 h, and then analyzed by Western blot.
  • (Ub)n polyubiquitin.
  • Fig. 3E depicts quantification of cell viability.
  • LN229 cells were transduced with the GIPZ lentiviral vectors that expressed either control (CT) shRNA or USP15 shRNAs clones 4 and 6 (shUSP15).
  • An equal number of cells (IxlO 6 cells in 10-cm plates) were then shifted to medium without glutamine in the presence (+) or absence (-) of the GS inhibitor MSO (2mM) for 5 days, after which cell viability was quantified by staining with 0.4% Trypan blue.
  • Cell viability was normalized to MSO-untreated control (-), which was set to 100%. Error bars represent the SEM of triplicates (P ⁇ 0001 by t test).
  • Fig. 3F is related to Fig. 3A.
  • Total ubiquitinated proteins were affinity-purified using TUBE2-agarose. Bound fractions were analyzed by SDS-PAGE and immunoblotting with anti-ubiquitin antibody.
  • Ub)n polyubiquitin.
  • Fig. 3G is related to Fig. 3B.
  • Total ubiquitinated proteins were affinity-purified using TUBE2-agarose. Bound fractions were analyzed by SDS-PAGE and immunoblotting with anti-ubiquitin antibody.
  • Ub)n polyubiquitin.
  • Fig. 3H is related to Fig. 3C.
  • Total ubiquitinated proteins were affinity-purified using TUBE2-agarose. Bound fractions were analyzed by SDS-PAGE and immunoblotting with anti-ubiquitin antibody.
  • Ub)n polyubiquitin.
  • Fig. 31 is related to Fig. 3D.
  • Total ubiquitinated proteins were affinity-purified using TUBE2-agarose. Bound fractions were analyzed by SDS-PAGE and immunoblotting with anti-ubiquitin antibody.
  • Ub)n polyubiquitin.
  • Fig. 3J depicts a Western blot.
  • Fig. 4A depicts a Western blot. USP15 +/+ and USP15 ⁇ !
  • ⁇ 293FT cells transfected with IKZF3TM (IKZF3 with a C-terminal Flag-Myc tag) plasmid for 48 h, were pre-treated with bortezomib (Bort, 1 pM) for 0.5 h, followed by lenalidomide (Len) treatment at 0 and 20 pM for 5 h.
  • Total ubiquitylated proteins were affinity -purified using TUBE2-agarose. Bound fractions and cell lysates (input) were analyzed by IB with indicated antibodies.
  • Ub)n polyubiquitin.
  • Fig. 4B depicts a Western blot.
  • Cells were pre-treated with Bort for 0.5 h, followed by Len treatment at 0, 10 and 20 pM for 5 h.
  • Total ubiquitylated proteins were affinity -purified using TUBE2-agarose.
  • Bound fractions and cell lysates (input) were analyzed by IB with indicated antibodies.
  • Ub)n polyubiquitin.
  • Fig. 4C depicts a Western blot.
  • Cells were pre-treated with CB-5083 (10 pM) for 0.5 h, followed by treatment with Len at 0, 10 and 20 pM for 5 h.
  • Total ubiquitylated proteins were affinity-purified using TUBE2-agarose. Bound fractions and cell lysates (input) were analyzed by IB with indicated antibodies.
  • Ub)n polyubiquitin.
  • Fig. 4D depicts a Western blot.
  • Cells were pre-treated with MG132 (20 pM) for 1 h, followed by treatment with CC-885 at 0, 1 and 10 pM for 3 h.
  • Total ubiquitylated proteins were affinity-purified using TUBE2-agarose. Bound fractions and cell lysates (input) were analyzed by IB with indicated antibodies.
  • Ub)n polyubiquitin.
  • Fig. 4E depicts a Western blot.
  • Cells were pre-treated with MG132 (20 pM) for 1 h, followed by treatment with dBETl, a potent BRD4 degrader (2 pM) for 4 h.
  • Total ubiquitylated proteins were affinity-purified using TUBE2-agarose. Bound fractions and cell lysates (input) were analyzed by IB with indicated antibodies.
  • Ub)n polyubiquitin.
  • Fig. 4F depicts a Western blot. USP15 + ' + and USP15 ⁇ ! ⁇ 293FT cells were pretreated with 10 pM Len for 0.5 h, followed by addition of CHX. Samples were harvested for IB analysis. The relative ratio of CKla:Actin was shown.
  • Fig. 4G depicts a Western blot. USP15 ⁇ 293FT cells, pre-treated with 10 pM CB- 5083, 20 pM MG132 and 2 pM MLN4924 for 0.5 h, were treated with 10 pM CC-885 for 3 h. Total ubiquitylated proteins were affinity-purified using TUBE2-agarose.
  • Fig. 4H depicts a Western blot related to Fig. 4A.
  • Total ubiquitinated proteins were affinity-purified using TUBE2-agarose. Bound fractions were analyzed by SDS-PAGE and immunoblotting with anti-ubiquitin antibody.
  • Ub)n polyubiquitin.
  • Fig. 41 depicts a Western blot related to Fig. 4B. Total ubiquitinated proteins were affinity-purified using TUBE2-agarose. Bound fractions were analyzed by SDS-PAGE and immunobl Otting with anti-ubiquitin antibody. (Ub)n: polyubiquitin.
  • Fig. 4J depicts a Western blot related to Fig. 4C. Total ubiquitinated proteins were affinity-purified using TUBE2-agarose. Bound fractions were analyzed by SDS-PAGE and immunoblotting with anti-ubiquitin antibody. (Ub)n: polyubiquitin.
  • Fig. 4K depicts a Western blot related to Fig. 4D.
  • Total ubiquitinated proteins were affinity-purified using TUBE2-agarose. Bound fractions were analyzed by SDS-PAGE and immunoblotting with anti-ubiquitin antibody.
  • Ub)n polyubiquitin.
  • Fig. 4L depicts a Western blot related to Fig. 4E.
  • Total ubiquitinated proteins were affinity-purified using TUBE2-agarose. Bound fractions were analyzed by SDS-PAGE and immunoblotting with anti-ubiquitin antibody.
  • Ub)n polyubiquitin.
  • Fig. 4M depicts a Western blot related to Fig. 4F.
  • Total ubiquitinated proteins were affinity-purified using TUBE2-agarose. Bound fractions were analyzed by SDS-PAGE and immunoblotting with anti-ubiquitin antibody.
  • Ub)n polyubiquitin.
  • Fig. 5A depicts a Western blot.
  • USP15 ⁇ ! ⁇ 293FT cells pre-treated with CB-5083 (10 pM) for 0.5 h, were induced with 20 pM lenalidomide for 5 h.
  • Total ubiquitylated proteins were affinity -purified using TUBE2-agarose beads.
  • Polyubiquitylated CKla proteins, pulled down with TUBE2 resin, were kept on ice as control (lane 1) or treated with or without recombinant USP15 (rUSP15) protein at 37°C for 0.5 h (lanes 2 & 3, respectively), followed by IB analysis.
  • CKla(Ub)n polyubiquitylated CKla.
  • Fig. 5B depicts a Western blot.
  • USPIS ⁇ 293FT cells pre-treated with CB-5083 for 0.5 h, were treated with CC-885 (10 pM) treatment for 4 h.
  • Total polyubiquitylated proteins pulled down with TUBE2 resin, were treated with or without recombinant USP15 (rUSP15) protein in the absence or presence of DUB inhibitors NEM (N-Ethylmaleimide; 20 mM) and PR- 619 (100 pM) at 37°C for 0.5 h, followed by SDS-PAGE analysis and immunoblotting with antibodies against GSPT1, ubiquitin and USP15.
  • GSPTl(Ub)n polyubiquitylated GSPT1.
  • Arrow indicates a native form of GSPT1, which was released after deubiquitylation of polyubiquitylated GSPT1 by rUSP15.
  • Fig. 5C depicts a Western blot.
  • SE and LE short and long exposures.
  • GSPTl(Ub)n polyubiquitylated GSPT1.
  • FIG. 5D depicts a Western blot.
  • IKZF3TM was purified from USPI5 ⁇ ! ⁇ 293FT cells.
  • An in vitro competitive ubiquitylation/deubiquitylation of IKZF3TM was carried out for Ih at 30°C in the presence or absence of El, E2, IIA Ub and recombinant CLR4 CRBN complex purified from insect cells in a final volume of 30 pl. Where indicated, Len (lenalidomide 50 pM) and recombinant USP15 (rUSP15) were added.
  • Fig. 5E depicts a Western blot related to Fig. 5 A.
  • Total ubiquitinated proteins were affinity-purified using TUBE2-agarose. Bound fractions were analyzed by SDS-PAGE and immunoblotting with anti-ubiquitin antibody.
  • Ub)n polyubiquitin.
  • Fig. 5F depicts a Western blot related to Fig. 5B.
  • Total ubiquitinated proteins were affinity-purified using TUBE2-agarose. Bound fractions were analyzed by SDS-PAGE and immunoblotting with anti-ubiquitin antibody.
  • Ub)n polyubiquitin.
  • Fig. 5G depicts a Western blot related to Fig. 5C.
  • Total ubiquitinated proteins were affinity-purified using TUBE2-agarose. Bound fractions were analyzed by SDS-PAGE and immunoblotting with anti-ubiquitin antibody.
  • Ub)n polyubiquitin.
  • Fig. 6A depicts a Western blot. USP15 was highly expressed in lenalidomide- resistant MM cell lines. Equal amounts of protein extracts from 3 lenalidomide (Len)-sensitive MM cell lines and 4 Len-resistant MM cell lines were analyzed by IB with the indicated antibodies. The relative ratios of USP15:Actin, CRBN:Actin and CUL4A:Actin, normalized to MM1S cells, are shown.
  • Fig. 6B depicts a Western blot. USP15-shRNA TRIPZ 2 RPMI8226 cells were induced with or without Dox (2 pg/ml) for 96 h. Cells were treated with 10 pM Len for 24 and 48 h.
  • FIG. 6C depicts a Western blot.
  • RPMI8226 cells stably expressing CT shRNA lentivirus (shCT) or GIPZ lentiviral shRNAs targeting human USP15 clones 4 or 6 (shUSP15_4 or 6), were treated with Len at 0, 1 and 10 pM for 48 h.
  • shCT CT shRNA lentivirus
  • shUSP15_4 or 6 GIPZ lentiviral shRNAs targeting human USP15 clones 4 or 6
  • Fig. 6D depicts a Western blot.
  • Fig. 6E depicts a Western blot.
  • Fig. 6F depicts cell viability demonstrating how depletion of USP15 sensitizes MM cells to lenalidomide.
  • RPMI8226 cells stably expressing Dox-inducible shRNA TRIPZ targeting USP15 clone 2 (USP75TRIPZ 2) were induced with or without Dox (2 pg/ml) for 3 days, and then treated with DMSO or 2, 10 pM Len for 5 days. Cell viability was quantified by staining with 0.4% Trypan blue. Cell viability was normalized to DMSO-treated control, which was set to 100%. Error bars represent the SEM of triplicates (P ⁇ 00001 by t test).
  • Fig. 6G depicts total cell number after different treatment conditions.
  • RPMI8226 cells were treated with DMSO (control: CT) or the DUB inhibitor PR-619 (1 pM) in the presence of lenalidomide (Len) at the indicated concentrations for 4 days.
  • Cell viability was quantified by staining with 0.4% Trypan blue. Error bars represent the SEM of triplicates (P ⁇ 003 by t test).
  • FIG. 6H depicts a Western blot demonstrating validation of USP15 depletion.
  • MM. IS cells were transduced with control shRNA lentivirus (CT), or GIPZ lentiviral shRNA particles targeting human USP15 by IB. [7&P75shRNA clones 4 and 6 with 80% knockdown efficiency were chosen for further analysis. Cell extracts were analyzed by immunoblotting with the indicated antibodies.
  • Fig. 61 depicts a Western blot.
  • MM IS cells, stably expressing control shRNA lentivirus (shCT) or GIPZ lentiviral shRNA targeting human USP15 clone 4 (shUSP15_4), were treated with Len at 0, 0.1 and 1 pM for 6 h.
  • Cell extracts were analyzed by immunoblotting with the indicated antibodies.
  • Fig. 6J depicts a Western blot.
  • U266 cells Dox-inducible USP75-shRNA TRIPZ 2
  • Cells were treated with Len at the indicated doses for 6 h.
  • Cell extracts were analyzed by immunoblotting with the indicated antibodies.
  • Fig. 6K depicts cell viability for vanous treatment conditions.
  • MM. IS cells, stably expressing control shRNA lentivirus (shCT) or GIPZ lentiviral shRNA targeting human USP15 clones 4 or 6 (shUSP15_4 or 6), were treated with DMSO or 2 pM Len for 5 days, after which cell viability was quantified by staining with 0.4% Trypan blue.
  • Fig. 6L depicts a Western blot.
  • Cell extracts were analyzed by SDS-PAGE and immunoblotting with indicated antibodies.
  • Fig. 7A depicts a Western blot. Glutamine-starved 293FT cells were treated 4 mM glutamine for 2 h.
  • Cell lysates were immunoprecipitated with CT IgG or USP15 antibody, and analyzed by IB with antibodies against GS, phosphorylated serine (P-Ser-USP15) or phosphorylated threonine (P-Thr-USP15).
  • the relative ratio of GS bound to USP15 was normalized to input GS.
  • the relative ratio of phosphorylated serine USP15 to USP15 input was normalized to untreated cells.
  • Fig. 7B depicts a Western blot. 293FT cells, transfected with CT or USP15 Flag plasmid for 24 h, were grown in complete medium (C; 2 mM glutamine) or starved of glutamine for 24 h, then treated with Torinl for 0.5 h, followed by 4 mM glutamine treatment for 1-2 h. Cell extracts were immunoprecipitated with anti-Flag. The relative ratios of phosphorylated serine USP15 Flag to total input USP15 Flag and endogenous GS interacted with USP15 Flag to input GS, normalized to that of untreated cells in lane 3, are shown.
  • Fig. 7C depicts a Western blot. /??7G/CshRNA TRIPZ 3 293FT cells, were induced with Dox for 24 h, and then transfected with CT or USP15 Flag plasmid. After 48 h, cell extracts were immunoprecipitated with anti-Flag and analyzed by IB with anti-phosphorylated serine. The relative ratio of phosphorylated serine USP15 Flag to USP1 Flag input is shown.
  • Fig. 8A depicts a Western blot. Glutamine-starved H1299 cells, pre-treated with 250 nM Torinl and 10 pM CB5083 for 0.5 h, were stimulated with 4 mM glutamine for 4 h. Cell extracts were analyzed by IB with the indicated antibodies. The relative ratio of GS : Actin is shown.
  • Fig. 8B depicts a Western blot. mTG/GshRNA TRIPZ 3 HEK293 cells were induced with or without Dox for 72 h. Cell extracts were analyzed by IB with the indicated antibodies. The relative ratio of GS:Actin is shown.
  • Fig. 8C depicts a Western blot. m/'G/CshRNA TRIPZ 3 293FT cells were induced with Dox for 72 h, and maintained in different glutamine concentrations for 24 h. Cell extracts were analyzed by IB with the indicated antibodies. The relative ratio of GS:Actin is shown.
  • Fig. 8D depicts a Western blot.
  • m7'(9/?-shRNA TRIPZ_3 293FT cells were induced with Dox for 72 h, and maintained in different glutamine concentrations for 24 h. Cells were treated with CHX for the indicated times. Cell extracts were analyzed by IB with the indicated antibodies. The relative ratio of GS:Actin is shown.
  • Fig. 8E depicts a Western blot. / «7G/?-shRNA TRIPZ 3 293FT cells were induced with Dox for 72 h, and maintained in different glutamine concentrations for 24 h. GS mRNA levels were quantified by qRT-PCR. Cell extracts were analyzed by IB with the indicated antibodies. The relative ratio of GS: Actin is shown.
  • Fig. 8F depicts a Western blot. mTO -shRNA TRIPZ 3 293FT cells were induced with Dox for 72 h, and maintained in different glutamine concentrations for 24 h. Cells were treated with different concentrations of MLN4924 for 14 h. Cell extracts were analyzed by IB with the indicated antibodies. The relative ratio of GS:Actin is shown.
  • Fig. 8G depicts a Western blot.
  • mTO7?-shRNA TRIPZ 3 293FT cells were induced with Dox for 72 h, and maintained in different glutamine concentrations for 24 h.
  • Cells were treated with different concentrations of MSO (G) for 14 h.
  • Cell extracts were analyzed by IB with the indicated antibodies. The relative ratio of GS:Actin is shown.
  • Fig. 9 depicts a schematic diagram of FL human USP15 protein. It contains DUSP domain, ubiquitin-like fold (UBL) and ubiquitin carboxyl-terminal hydrolase (UCH).
  • UDL ubiquitin-like fold
  • UCH ubiquitin carboxyl-terminal hydrolase
  • Fig. 10A depicts a mass spectrum of a USP15 peptide containing phosphorylated S229.
  • Fig. 10B depicts a putative phosphorylation site at serine 229, located in the linker between the UBL domain and UCH domain, is conserved among mammalian USP15 orthologs.
  • Fig. 10C depicts he phosphorylation peptide abundance calculated by phosphorylated peptides/ (phosphorylated peptides + unmodified peptides)* 100%.
  • Fig. 11 depicts a proposed model where USP15 is a key regulator of the CRL4 CRBN - USP15 pathway to control the stability of natural substrate GS and neo-substrates.
  • RNAi RNA interference
  • CRISPR/Cas9 gene editing in vivo and in vitro biochemical approaches show that USP15 is a key regulator of the CRL4 CRBN - USP15 pathway (historically referred to as the 'CRL4 CRBN -p97 pathway 1 ) to control the stability of glutamine synthetase (GS) and neo-substrates, including IKZF1/3, CKla, RNF166, GSPT1 and BRD4 (a target of CRBN-based PROTAC dBETl), all of which are crucial drug targets in many cancers.
  • GS glutamine synthetase
  • neo-substrates including IKZF1/3, CKla, RNF166, GSPT1 and BRD4 (a target of CRBN-based PROTAC dBETl), all of which are crucial drug targets in many cancers.
  • USP15 antagonizes ubiquitylation of CRL4 CRBN target proteins, thereby preventing their degradation. Notably, USP15 is highly expressed in IMiD-resistant cell lines, and depletion of USP15 sensitizes these cells to lenalidomide.
  • USP15 protein expression can be used as a predictive biomarker of response or resistance to IMiD therapy in patients with MM and also advance the development of a class of drugs to degrade pathogenic proteins in cancer via molecular glue degraders (CRBN E3 ligase modulators: CELMoDs) and proteolysis-targeting chimaeras (PROTACs).
  • Inhibition of USP15 represents a valuable therapeutic opportunity to potentiate IMiD/CELMoD and PROTAC therapies for the treatment of cancer (such as glioblastoma (GBM), MM, Myelodysplastic syndrome with deletion 5q (Del(5q)MDS) and acute myeloid leukemia (AML)) and other human diseases.
  • GBM glioblastoma
  • MM Myelodysplastic syndrome with deletion 5q
  • AML acute myeloid leukemia
  • the USP15 inhibitor is selected from USP15 anti sense mRNA, USP15 siRNA, USP15 shRNA, USP15 miRNA, USP15 oligonucleotides and chemical inhibitors of USP15 such as PR-619. These USP15 inhibitors can further be used in combination with IMiDs (now called CELMoDs including thalidomide, lenalidomide, pomalidomide, CC-122, CC-220 and CC-885) or CRBN-based PROTACs (dBETl) to treat cancer.
  • IMiDs now called CELMoDs including thalidomide, lenalidomide, pomalidomide, CC-122, CC-220 and CC-885
  • CRBN-based PROTACs CRBN-based PROTACs
  • the present disclosure is directed to methods of treating cancer comprising administering a therapeutically effective amount of a USP15 inhibitor and at least one chemotherapeutic drug to a subject in need thereof.
  • the chemotherapeutic drug can be an immunomodulatory drug (IMiD), cereblon E3 ligase modulator (CELMoD), or CRBN-based proteolysis-targeting chimaera (PROTAC).
  • IMD immunomodulatory drug
  • CELMoD cereblon E3 ligase modulator
  • PROTAC CRBN-based proteolysis-targeting chimaera
  • the chemotherapeutic drug can be selected from the group consisting of thalidomide, lenalidomide, pomalidomide, CC-122, CC-220, and CC-885.
  • the CRBN-based PROTAC can be selected from the group consisting of ARV-825 (2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2- f][l,2,4]triazolo[4,3-a][l,4]diazepin-6-yl)-N-(4-(2-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-l,3- dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)ethoxy)phenyl)acetamide), dBETl ((6S)-4-(4- Chlorophenyl)-N-[4-[[2-[[2-(2,6-dioxo-3-piperidinyl)-2,3-dihydro-l,3-dioxo-lH-isoindol-4- yl
  • the cancer can be multiple myeloma, glioblastoma, deletion 5q subtype of myelodysplastic syndrome, or acute myeloblastic leukemia.
  • the cancer can be IMiD-resistant.
  • the disclosure is further directed to methods of diagnosing and treating resistance to IMiD therapy in a subject with multiple myeloma undergoing IMiD therapy, the method comprising: a) measuring an amount of USP15 protein in the subject; b) comparing the amount of USP15 protein in the subject to a reference; c) diagnosing the subject with resistance to IMiD therapy if the amount of USP15 protein in the subject is higher than the reference; and d) administering a USP15 inhibitor to the subject.
  • Measuring the amount of USP15 protein in the subject can comprise using immunohistochemical (IHC) staining of USP15 protein in bone marrow sections of the subject.
  • the reference can be obtained from measuring an amount of USP15 protein from a different subject or subjects without multiple myeloma.
  • the USP15 inhibitor can be a nucleic acid or a chemical inhibitor.
  • the nucleic acid can be selected from the group consisting of USP15 antisense mRNA,USP15siRNA,USP15shRNA, USP15 miRNA, andUSPlS oligonucleotides.
  • the chemical inhibitor can be a deubiquitinating enzyme (DUB) inhibitor.
  • the chemical inhibitor can be selected from the group consisting of PR-619, NSC632839, and N-Ethylmaleimide (NEM).
  • the chemical inhibitor can be mitoxantrone or an ubiquitin variant.
  • the subject can be a mammal.
  • the subject can also be a human, mouse, or rat.
  • Example 1 USP15 is a GS-interacting partner
  • RNA interference RNA interference
  • CRISPR/Cas9 gene editing approaches A previous study using stable isotope labeling of amino acids in cell culture (SILAC)-based quantitative MS to characterize changes in the ubiquitinome of Jurkat cells treated with the proteasome inhibitor MG-132 or the DUB inhibitor PR-619 for 4 h revealed that while increased ubiquitylation of GS was observed in cells treated with MG132, PR-619 treatment decreased the ubiquitination of GS, suggesting that GS is rapidly ubiquitylated and degraded in PR-619-treated cells, leading to a decrease in abundance of both native and ubiquitylated forms of GS.
  • SILAC stable isotope labeling of amino acids in cell culture
  • the USP15-dependent degradation of GS is a post-translational regulation because the mRNA level of GS remained unchanged in USP15-KO 293FT cells (Fig. 2L).
  • Fig. 2D-2F the downregulation of endogenous GS protein levels observed in USP15-K0 293FT cells was rescued by blocking GS ubiquitylation using MLN4924 or the GS inhibitor MSO in a prior study, indicating that USP15 controls GS degradation in downstream of CRL4 CRBN .
  • Example 3 USP15 deubiquitylates polvubiauitylated GS in vivo and in vitro
  • GBM is the most frequent adult primary malignant brain tumors, and it is the second leading cause of cancer mortality in adults under 35 years of age. GBM and many other tumors expressing high GS levels can synthesize glutamine de novo, grow and proliferate in the absence of exogenous glutamine. Interestingly, USP15 amplification confers poor prognosis in patients with GBM. The above data provide compelling evidence that USP15 is a key component of CRL4 CRBN -p97 pathway to regulate GS stability. It was found that depletion of USP15 by shRNAs in LN229 cells resulted in a marked reduction in the steady-state level of GS (Fig. 3J).
  • Example 4 USP15 regulates CELMoD-induced ubiquitylation and degradation of neo-substrates
  • Example 5 USP15 deubiquitylates poly ubiquity lated neo-substrates in vitro
  • USP15 is a deubiquitylating enzyme, it was next tested whether it could remove polyubiquitin chains from neo-substrates of CRL4 CRBN in vitro.
  • ubiquitin-binding TUBE2 resin was utilized to purify polyubiquitylated neo-substrates from cellular extracts of USP15-KO cells treated with the CELMoDs lenalidomide and CC-885 in the presence of p97 or proteasome inhibitor, and then performed the in vitro deubiquitylation assays using rUSP15.
  • USP15 directly deubiquitylates polyubiquitylated CKla forms (Fig. 5A and 5E) and GSTP1 (Fig.
  • the ‘competitive’ ubiquitylation/de-ubiquitylation assay that contains all key components of the UPS, including recombinant El, E2, CRL4 CRBN , neo-substrate IKZF3 FM (FM: C-terminal Flag- Myc) purified from USP15 ⁇ 293FT cells and rUSP15 was performed in the presence or absence of lenalidomide to mimic the biological properties of in vivo cells.
  • the in vitro competitive ubiquitylation/deubiquitylation assay strongly indicated that lenalidomide promoted CRL4 CRBN - mediated ubiquitylation of IKZF3 in vitro in an IMiD-dependent manner (Fig.
  • Example 6 Depletion of USP15 promotes IMiD-induced degradation of IKZF1/3 in lenalidomide- resistant MM cell lines
  • Glutamine activates the highly conserved, atypical Serine/Threonine kinase mammalian Target of Rapamycin Complex 1 (mTORCl), which regulates protein translation, cell growth and autophagy.
  • the core components of mTORCl consist of mTOR, Raptor, and mLST8.
  • Glutamine may directly activate USP15 through mTORCl.
  • IP experiments were used to determine that glutamine significantly stimulated the binding of endogenous GS to endogenous USP15, and correlated with enhanced serine-phosphorylated, but not threonine-phosphorylated USP15 (Fig. 7A).
  • mTOR As shown in Fig. 8A, inhibition of mTOR promoted glutamine-induced GS degradation, which was blunted by CB5083, indicating that mTOR functions downstream of p97 to regulate GS stability. Consistent with these results, a recent study reported that mTOR regulates GS degradation in a proteasome-dependent manner through an unknown mechanism.
  • Dox-inducible shRNA was used in TRIPZ system to deplete mTOR in HEK293 and 293FT cells. Depletion of mTOR by shRNA promoted GS degradation (Fig. 8B-8D) while the mRNA levels of GS remained unchanged in mTOR knockdown cells (Fig. 8E).
  • USP15 Flag was immune-purified from 293FT cells treated with DMSO or 250 nM Torinl for 16 h and analyzed by MS analysis.
  • Known domains of USP15 are shown in Fig. 9.
  • S229 was of particular interest, because the proteomic studies revealed that S229 was phosphorylated (Fig. 10A).
  • Fig. 10A it represents an mTOR consensus sequence motif S/T-P (where S is serine; T is threonine; and P is proline) (Fig.
  • the CRL4 CRBN -p97 pathway is hereinafter called the CRL4 CRBN -USP15 pathway.
  • FIG. 11 A proposed model for the role of USP15 in regulating the stability of natural substrate GS and neo-substrates in the CRL4 CRBN -USP15 pathway is illustrated in Fig. 11.
  • High glutamine induces GS acetylation at lysines 11 and 14 to create a degron that binds CRBN.
  • acetylated GS is ubiquitylated by CRL4 CRBN , segregated by p97 and degraded by the proteasome.
  • GBM glioblastoma
  • upregulation of USP15 stabilizes GS, a key metabolic enzyme in glutamine metabolism, which is essential for tumor growth.
  • USP15 antagonizes CELMoD/PROTAC-induced ubiquitylation of target proteins, thereby inhibiting their subsequent degradation. This accounts for intrinsic resistance in IMiD-resistant MM cell lines that greatly overproduce USP15 protein. Inhibition of USP15 represents a valuable therapeutic target to potentiate CELMoD and CRBN-based PROTAC therapies for the treatment of cancer including MM, deletion 5q (del(5q)) subtype of myelodysplastic syndrome (MDS), acute myeloblastic leukemia (AML) and other human diseases.
  • MDS myelodysplastic syndrome
  • AML acute myeloblastic leukemia
  • USP15 was identified as a GS -interacting partner based on two independent proteomic studies.
  • the presented results provide the first direct evidence that USP15 deubiquitylates and stabilizes seven tested CRL4 CRBN protein targets, including GS, IKZF1, IKZF3, CKla, RNF166, GSPT1 and BRD4 via natural substrate, molecular glue and PROTAC mechanisms. It functions downstream of CRL4 CRBN and upstream of p97 and proteasome. This accounts for intrinsic resistance of IMiDs in MM.
  • CRL4 CRBN a potential biomarker of response or resistance to IMiD therapy in patients with MM.
  • USP15 was identified as a potential GS -interacting protein by two independent proteomic studies. Preliminary results demonstrated that endogenous USP15 interacts with endogenous GS, and deubiquitylates polyubiquitylated GS in vivo and in vitro (Fig. 6A-6L). Depletion of USP15 by the DUB inhibitors (Fig. 2A-2L), shRNA-mediated knockdow n (Fig. 3A- 3J), or CRISPR/Cas9 genome editing promotes GS ubiquitylation and subsequent degradation (Fig. 4A-4M, 5A-5G, and 6A-6L). The sequential events of GS ubiquitylation and degradation were defined, and it was found that USP15 acts downstream of CRL4 CRBN and upstream of p97 and proteasome by counteracting the function of CRL4 CRBN to control GS stability.
  • HEK293 cells, 293FT cells, H1299 cells, MCF7 cells, MM. IS, U266, NCI-H929 and RPMI-8226 were purchased from ATCC (American Type Culture Collection, Manassas, VA, USA). KMS-11, ARP-1 and OPM-1 cell lines were provided by from a lab at the University of Pennsylvania, Philadelphia, PA, USA.
  • Anti-glutamine synthetase (E-4; sc-74430), anti-IKZFl (E-2, sc-398265), anti- IKZF3/Aiolos (3H5-G7; sc-293421) antibodies were from Santa Cruz Biotechnology.
  • Mouse monoclonal anti-USP15 antibody (1C10, H00009958-M01) used for Western blot was from Abnova.
  • Anti-ubiquitin (P4D1-A11, 05-944), Anti-CRBN (HPA045910), anti-Flag HRP, and anti-Myc HRP antibodies were from Sigma.
  • Anti-p97/VCP (abll433), anti-GSPTl (ab49878), anti-DDBl (ab21080), anti-ty Actin HRP conjugated (AC-15, ab49900) antibodies were from Abeam.
  • Anti-CUL4A (2699) antibody was from Cell Signaling Technology.
  • Anti-BRD4 Antibody (BL-149-2H5; A700-004) was from Bethyl.
  • Anti-Flag HRP conjugated antibody 600- 403-383 was from Rockland Immunochemicals.
  • HRP Goat Anti-Rabbit IgG PI-1000
  • HRP Horse Anti-Mouse IgG PI-2000
  • IP immunoprecipitation
  • rabbit polyclonal anti-USP15 antibody (NB110- 4069) was fromNovus Biologicals.
  • Rabbit IgG Isotype Control normal rabbit IgG; sc-2027 was from Santa Cruz Biotechnology.
  • EZview Red anti-Flag M2 (F2426) and EZview Red Anti-c-Myc Affinity Gels (E6654) were from Sigma.
  • Human USP15 expression vector pcDNA3.1+/C-(K)-DYK with a C-terminal Flag tag, expressing USP15 isoform 1 (NM_001252078.2) was purchased from GenScript.
  • Human GS expression vector pCMV6-GS-Myc-Flag (C-terminal Myc-Flag tag) were purchased from OriGene.
  • pCMV6-GS-Myc was generated by introducing a STOP codon between Myc and Flag by using a QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA).
  • the full-length cDNA of IKZF3 was amplified from the IKZF3 expression vector pcDNA3.2 with a N-terminal HA tag (a kind gift from Dr. Benjamin Ebert, Dana Farber Cancer Institute), and then subcloned into pCMV6-Flag-Myc tags (pCMV6-IKZF3Flag-Myc). All cDNAs cloned into mammalian expression vectors are confirmed by DNA sequencing.
  • the TRIPZ inducible lentiviral shRNAs for human USP15 (clone ID #1: V2THS_5710; clone ID #2: V2THSJ3437) were purchased from Horizon Discovery. The shRNAs were induced by using 2 pg/ml doxycycline for 3-4 days.
  • GIPZ Lentiviral shRNA the GIPZ lentiviral constructs expressing nontargeting (control, CT) and human USP15 shRNAs (USP15_1 shRNA: V2LHS_196921; USP15 2 shRNA: V2LHS_5710; USP15_3 shRNA: V2LHS 3436; USP15_4 shRNA: V2LHS 13437; USP15_5 shRNA: V3LHS_336555; and USP15_6 shRNA: V3LHS_336551) were purchased from Horizon Discovery.
  • Six lentiviruses in the GIPZ Lentiviral shRNA vectors targeting USP15 were screened to identify shRNAs that optimally suppressed USP15.
  • Virus preparation and cell infection were performed according to the manufacturer’s protocol, with minor modifications. Briefly, shRNA-encoding plasmids were co-transfected with psPAX2 (packaging plasmid) and pMD.2G (enveloping plasmid) into HEK293FT cells using Fugene HD (Promega). Virus -containing supernatants were harvested at 48 h and 72 h post transfection. The lentiviruses were precipitated using PEG-it virus precipitation solution according to the manufacturer’s protocol (System Biosciences).
  • Target cells were transduced with lentiviral particles in the presence of 8 pg/ml polybrene, followed by selection with puromycin (1 pg/ml) for multiple myeloma cell lines, and 2-4 pg/ml for 293 cells and 293FT cells) for 2 weeks. Knockdown efficiencies were analyzed by immunoblot. Generation of USP15 knockout 293FT cells by CRISPR genome editing
  • HEK293FT cells were cultured in 10-cm plates. Cells were starved of glutamine for 24h and pretreated with the inhibitors before adding Q4 for 2h. Cells were harvested by washing in cold PBS 2x and freeze down at -80°C. Cells were lysed with 1ml BD150 (10 mM Tris [pH 7.5], 150 mM NaCl, 1% Triton X-100, and 1 mM DTT containing a protease inhibitor cocktail).
  • Antibodies used for the IP include rabbit polyclonal anti- USP15 Ab (1 ig/pil) using 4 pl and normal rabbit IgG control using 10 pl (0.4 pg/pl), which is an unconjugated, affinity purified isotype control immunoglobulin from rabbit in PBS with 0.1% sodium azide and 0.1% gelatin.
  • IP occurred for 2 hr. Then, 30 pl protein G beads were added for 1 h. IP buffer was used 3x, and elution occurred with 50 pl 1.5x SDS-SB.
  • LN-229 cell lines were seeded in eight to ten 10-cm cell culture plates and cultured until they reached 80% confluence. Cells were washed with lx PBS and lysed in 500pl 1%NP4O lysis buffer (25mM Tris pH 7.5, 150mM NaCl, 1% NP40, 0.5mM, phenylmethylsulfonylfluoride (PMSF), protease inhibitor cocktail (Roche)). Lysates were centrifuged at 13000 rpm for 10 minutes. Protein concentration was measured using Bradford assay (Bio- Rad Laboratories). For the first IP MS experiment 3 mg of protein was used and for the second 5 mg.
  • 1%NP4O lysis buffer 25mM Tris pH 7.5, 150mM NaCl, 1% NP40, 0.5mM, phenylmethylsulfonylfluoride (PMSF), protease inhibitor cocktail (Roche)
  • Lysates were centrifuged at 13000 rpm for 10
  • the concentration of the USP15 antibody (NB 110-40690, Novus Biologicals, UK) and of the normal Rabbit IgG control (12-370, Millipore, Germany) was 5pg/mg of lysate.
  • the mixture of antibody /lysate was left rotating gently at 4°C overnight.
  • the beads containing polyubiquitylated proteins were mixed in 30 pl of ubiquitylation buffer, followed by addition of 1 pg recombinant USP15 (rUSP15) protein (catalog# E-594-050, R&D Systems) and incubated at 37°C for 0.5 h.
  • rUSP15-treated samples were mixed with 30 pl of 2xSDS sample buffer, boiled for 5 min and subjected to Western blot analysis.
  • IKZF3TM was purified from USP15 ⁇ 293FT cells.
  • An in vitro competitive ubiquitylation/deubiquitylation of IKZF3 FM was carried out for Ih at 30°C in the presence or absence of El, E2, I IA Ub and recombinant CLR4 CRBN complex purified from insect cells in a final volume of 30 pl in the presence of 50 pM lenalidomide and recombinant USP 15 (rUSP15; 1 pg) were added. Reactions were stopped by mixing with equal amount of 2xSDS sample buffer and analyzed by SDS-PAGE and immunoblotting with antibodies against Flag, USP 15 and CRBN.

Abstract

La présente invention concerne de manière générale des compositions et des méthodes associées à des inhibiteurs d'USP15. Plus particulièrement, la présente invention concerne des méthodes de régulation de la voie CRL4CRBN-USP15 (appelée précédemment voie CRL4CRBN-p97) et des niveaux de glutamine synthétase et des méthodes de traitement de maladies telles que le cancer par l'administration d'inhibiteurs d'USP15.
PCT/US2022/076022 2021-09-08 2022-09-07 Méthodes d'utilisation d'inhibiteurs d'usp15 WO2023039405A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202163241555P 2021-09-08 2021-09-08
US63/241,555 2021-09-08

Publications (1)

Publication Number Publication Date
WO2023039405A1 true WO2023039405A1 (fr) 2023-03-16

Family

ID=85507686

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2022/076022 WO2023039405A1 (fr) 2021-09-08 2022-09-07 Méthodes d'utilisation d'inhibiteurs d'usp15

Country Status (1)

Country Link
WO (1) WO2023039405A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150344887A1 (en) * 2014-05-29 2015-12-03 Korea Institute Of Science And Technology siRNA FOR INHIBITION OF USP15 EXPRESSION AND PHARMACEUTICAL COMPOSITION CONTAINING THE SAME
WO2017117006A1 (fr) * 2015-12-29 2017-07-06 The Board Of Trustees Of The Leland Stanford Junior University Utilisation d'un inhibiteur de dhodh en association avec un inhibiteur de récupération pyrimidique
US20190004033A1 (en) * 2013-12-06 2019-01-03 Celgene Corporation Methods for determining drug efficacy for the treatment of diffuse large b-cell lymphoma, multiple myeloma, and myeloid cancers

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190004033A1 (en) * 2013-12-06 2019-01-03 Celgene Corporation Methods for determining drug efficacy for the treatment of diffuse large b-cell lymphoma, multiple myeloma, and myeloid cancers
US20150344887A1 (en) * 2014-05-29 2015-12-03 Korea Institute Of Science And Technology siRNA FOR INHIBITION OF USP15 EXPRESSION AND PHARMACEUTICAL COMPOSITION CONTAINING THE SAME
WO2017117006A1 (fr) * 2015-12-29 2017-07-06 The Board Of Trustees Of The Leland Stanford Junior University Utilisation d'un inhibiteur de dhodh en association avec un inhibiteur de récupération pyrimidique

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DANIEL J. DEANGELO; ANDREW M. BRUNNER; LILLIAN WERNER; DAVID AVIGAN; AMIR T. FATHI; ADAM S. SPERLING; ABIGAIL WASHINGTON; DINA STR: "A phase I study of lenalidomide plus chemotherapy with mitoxantrone, etoposide, and cytarabine for the reinduction of patients with acute myeloid leukemia", AMERICAN JOURNAL OF HEMATOLOGY, NEW YORK, NY, US, vol. 93, no. 2, 21 November 2017 (2017-11-21), US , pages 254 - 261, XP071632032, ISSN: 0361-8609, DOI: 10.1002/ajh.24968 *
NGUYEN THANG VAN: "USP15 antagonizes CRL4 CRBN -mediated ubiquitylation of glutamine synthetase and neosubstrates", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF SCIENCES, vol. 118, no. 40, 5 October 2021 (2021-10-05), XP093046812, ISSN: 0027-8424, DOI: 10.1073/pnas.2111391118 *
ZHOU LILI, JIANG HUA, DU JUAN, LI LU, LI RONG, LU JING, FU WEIJUN, HOU JIAN: "USP15 inhibits multiple myeloma cell apoptosis through activating a feedback loop with the transcription factor NF-κBp65", EXPERIMENTAL AND MOLECULAR MEDICINE, SEOUL, KR, vol. 50, no. 11, 1 November 2018 (2018-11-01), KR , pages 1 - 12, XP093046810, ISSN: 1226-3613, DOI: 10.1038/s12276-018-0180-4 *

Similar Documents

Publication Publication Date Title
Bury et al. NFE2L3 controls colon cancer cell growth through regulation of DUX4, a CDK1 inhibitor
Tsofack et al. NONO and RALY proteins are required for YB-1 oxaliplatin induced resistance in colon adenocarcinoma cell lines
Mondello et al. Dual inhibition of histone deacetylases and phosphoinositide 3-kinase enhances therapeutic activity against B cell lymphoma
Hu et al. The USP10-HDAC6 axis confers cisplatin resistance in non-small cell lung cancer lacking wild-type p53
Ganguly et al. Mucin 5ac serves as the nexus for β-catenin/c-myc interplay to promote glutamine dependency during pancreatic cancer chemoresistance
Hsu et al. Signal peptide peptidase promotes tumor progression via facilitating FKBP8 degradation
Liu et al. JQ1, a BET‐bromodomain inhibitor, inhibits human cancer growth and suppresses PD‐L1 expression
Pei et al. Piperlongumine conjugates induce targeted protein degradation
Yang et al. Regulation of VKORC1L1 is critical for p53-mediated tumor suppression through vitamin K metabolism
Wang et al. CYLD deficiency enhances metabolic reprogramming and tumor progression in nasopharyngeal carcinoma via PFKFB3
Park et al. Lipid raft‐disrupting miltefosine preferentially induces the death of colorectal cancer stem‐like cells
Ali et al. The mTORC1-SLC4A7 axis stimulates bicarbonate import to enhance de novo nucleotide synthesis
Chen et al. Targeting fatty acid synthase sensitizes human nasopharyngeal carcinoma cells to radiation via downregulating frizzled class receptor 10
Yao et al. Cyclin K interacts with β-catenin to induce Cyclin D1 expression and facilitates tumorigenesis and radioresistance in lung cancer
US20220016130A1 (en) Methods and materials for identifying and treating bet inhibitor-resistant cancers
Liu et al. Hypoxia-induced proteasomal degradation of DBC1 by SIAH2 in breast cancer progression
Yang et al. CircNFATC3 promotes the proliferation of gastric cancer through binding to IGF2BP3 and restricting its ubiquitination to enhance CCND1 mRNA stability
Noh et al. Additive effect of Cd73 inhibitor in colorectal cancer treatment with cdk4/6 inhibitor through regulation of Pd-L1
De Martino et al. Radiation therapy promotes unsaturated fatty acids to maintain survival of glioblastoma
Shin et al. Targeting FLT3-TAZ signaling to suppress drug resistance in blast phase chronic myeloid leukemia
Ming-Ming et al. Polyphyllin I promotes cell death via suppressing UPR-mediated CHOP ubiquitination and degradation in non-small cell lung cancer
Seefried et al. Nuclear AREG affects a low‐proliferative phenotype and contributes to drug resistance of melanoma
WO2023039405A1 (fr) Méthodes d'utilisation d'inhibiteurs d'usp15
Du et al. Bi-steric mTORC1 inhibitors induce apoptotic cell death in tumor models with hyperactivated mTORC1
Gan et al. Research progress on Mono-ADP-Ribosyltransferases in human cell biology

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22868261

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 18690470

Country of ref document: US

NENP Non-entry into the national phase

Ref country code: DE